Peripheral blood in the circulatory system of a human is comprised principally of red blood cells, platelets, and white blood cells or leukocytes. The family of white blood cells is comprised of lymphocytes, neutrophils, monocytes, eosinophils, and basophils. Lymphocytes are of T cell or B cell subtypes primarily; additional subsets of lymphocytes are known also. The variety of functions of leukocytes and their clinical relevance has generated great interest in the scientific community.
Neutrophils, eosinophils, and basophils are identified as "granulocytes" because of their content of cytoplasmic granules. Granulocytes and monocytes are identified as "phagocytes" because of their ability to phagocytose or ingest bacteria, other micro-organisms and other types of foreign materials referred to generally as "antigens". This phagocytic function is important in defending the host against a variety of infections and further, is important in various types of inflammatory disorders. Phagocytes are produced from common progenitor cells in the bone marrow circulate in the peripheral blood and finally, enter tissues as necessary for control of infection or for participating in an inflammatory reaction. Such response functions are found in human, and animal phagocytes, i.e., in primates and canines.
The neutrophil is the most common leukocyte in human and animal peripheral blood. One microliter of normal human whole blood includes, on average, 5 .times. 10.sup.3 leukocytes, of which 3,975 are neutrophils, 150 are eosinophils, 25 are basophils, 250 are monocytes, and 1,500 are lymphocytes.
In the immunological response of granulocytes or monocytes to any type of infection or inflammation, these cells are first activated to migrate to the appropriate area in response to "chemo-attractant factors" such as certain bacterial products, complement components, etc. This attraction process is termed "chemotaxis". Once in an area of inflammation or infection, granulocytes and monocytes then undertake to establish a firm attachment to their targets. For this purpose, these cells possess a number of specific cell surface receptor glycoproteins that promote this interaction, such as complement, Fc, and fibronectin receptors.
One of the most important family of cell surface receptor glycoproteins involved in phagocyte adhesion is the leukocyte cell adhesion molecule family identified as LEU-CAM or CD11/CD18. This family is comprised of at least three (3) cell surface proteins which have two (2) subunits each. They share a common beta subunit, CD18, of 94,000 daltons molecular weight and have different alpha subunits. The known members of this family are termed LFA-1 (CD11a/CD18), Mol (CD11b/CD18), and P150,94 (CD11c/CD18). These glycoproteins have alpha subunits of 180,000, 155,000 and 150,000 dalton molecular weight, respectively. Each of these cell surface proteins have been specifically identified through the use of monoclonal antibodies. The biological importance of this family of surface glycoproteins has been recognized through the identification of a human disease in which leukocytes are genetically deficient in this family of antigens. The disease is characterized by recurrent severe bacterial infections and deficiencies in adhesion-dependent functions, such as, phagocytosis, chemotaxis, leukoaggregation, and neutrophil spreading on plastic.
Mol is a cell surface glycoprotein present on granulocytes, mononuclear phagocytes and null cells (Todd, R.F. III, Nadler, L.M., and Schlossman, S.F., Antigens on human monocytes, Journal of Immunology. 126:1435-1442, 1981). In humans, this molecule consists of two non-covalently linked proteins of 155,000 and 94,000 daltons (Todd, R.F. III, van Agthoven, A., Schlossman, S.F., and Terhorst, D., Structure analysis of differentiation antigens Mol and Mo2 on human monocytes, Hybridoma 1:329-337, 1982). This complex has been shown to mediate cell adhesion to a variety of surfaces, including other granulocytes, endothelium, and inert substrates. Genetic deficiencies in these molecules result in recurrent bacterial infections due to the inability of granulocytes to mediate an anti-microbial inflammatory response. Patients who are deficient in these molecules are characterized by an elevated leukocyte count (called "leukocytosis") and functional defects in phagocyte activity as measured in vitro by reduced or absence of aggregation adhesion to substrates, chemotaxis, and phagocytosis of opsonized particles. Activation of granulocytes and monocytes by soluble inflammatory mediators increases expression of these molecules (Todd. R.F. III, Arnaout, M.A., Rosin, R.E., Crowley, C.A., Peters, W.A., and Babior, B.M. The subcellular localization of Mol (Mol alpha; formerly gp .sup.110), a surface of glycoprotein associated with neutrophil adhesion, J. Clin. Invest., 74:1280-1290, 1984; Arnaout, M.A., Hakim, R.M. Todd, R.F., Dana, N. and Colten, H.R. Increased expression of an adhesion-promotion surface glycoprotein in the granulocytopenia of hemodialysis. New Engl. J. Med., 312:457-462, 1985). Monoclonal antibodies directed against the Mol glycoprotein effectively prevent neutrophil aggregation in vitro as well as prevent phagocytosis.
The Mol glycoprotein has been of particular interest because its molecular structure has the capacity to bind a component termed "iC3b", a fragment of the third component of complement (Arnaout, M.A., Todd, R.F. III, Dana N., Melamed, J., Schlossman, S.F., and Colten, H.R. Inhibition of phagocytosis of complement C3 or IgG-coated particles and of iC3b binding by monoclonal antibodies to a monocyte-granulocyte membrane glycoprotein (Mol), J. Clin. Invest., 72:171-179, 1983. Also, the Mol glycoprotein is critically important in all of the adhesion-dependent phagocyte functions. Different monoclonal antibodies have been shown to inhibit the functions of the Mol glycoprotein.
The monoclonal antibody derived from the novel hybrid cell line embodying the invention is capable of inhibiting the adhesion-dependent function of, but does not bind the iC3b fragment of the third component of complement. This monoclonal antibody is identified by the designation "MY904". It binds to specific antigenic sites of neutrophils, i.e. to the antigenic site on the CD11b/CD phagocyte surface protein which is specifically involved in granulocyte adhesion. The addition of the MY904 monoclonal antibody to phagocytes will thus inhibit adhesion-dependent phagocyte functions; but it does not inhibit other functions of either the CD11b/CD18 molecule, such as binding of complement component iC3b, or other types of neutrophil or monocyte functions, such as Fc receptor activation, activation of the respiratory burst by chemotactic peptide or phorbol diester and others.
The utility for such a site specific monoclonal antibody is very diversified. The binding of the MYg04 monoclonal antibody to the adhesion dependent domain of neutrophils could specifically inhibit neutrophil migration to an area of inflammation or infection. In addition, such binding to neutrophils could inhibit adhesion and spreading of activated neutrophils already within such an inflammatory or infected site, thus blocking the release of toxic substances by the neutrophils. The monoclonal antibody MY904 could be labelled with a suitable marker for immunoassay of the CD11b/CD18 molecule or be conjugated to a suitable substrate for depletion of bound cells by fluorescence activated cell sorting or magnetic bead separation. The ability of the MY904 monoclonal antibody to block certain phagocyte functions would have special utility for in depth studY of phagocyte function, especially where excess or harmful phagocyte function is involved in clinical disorder. Further, this monoclonal antibody is useful for quantitating surface expression of CD11b/CD18 and thereby can be applied to diagnose the Mol deficiency disease described herein.